Glycerophospholipids, the main component of biological membranes, contain a glycerol core with fatty acids attached as R groups at the sn-1 position and sn-2 position, and a polar head group joined at the sn-3 position via a phosphodiester bond. The specific polar head group (e.g., phosphatidic acid, chlorine, ethanolamine, glycerol, inositol, serine, cardiolipin) determines the name given to a particular glycerophospholipid, thus resulting in phosphatidylcholines [“PC”], phosphatidylethanolamines [“PE”], phosphatidylglycerols [“PG”], phosphatidylinositols [“PI”], phosphatidylserines [“PS”] and cardiolipins [“CL”]. Glycerophospholipids possess tremendous diversity, not only resulting from variable phosphoryl head groups, but also as a result of differing chain lengths and degrees of saturation of their fatty acids. Generally, saturated and monounsaturated fatty acids are esterified at the sn-1 position, while polyunsaturated fatty acids are esterified at the sn-2 position.
Glycerophospholipid biosynthesis is complex. Table 1 below summarizes the steps in the de novo pathway, originally described by Kennedy and Weiss (J. Biol. Chem., 222.193-214 (1956)):
TABLE 1General Reactions Of de Novo Glycerophospholipid Biosynthesissn-Glycerol-3-PhosphateGlycerol-3-phosphate acyltransferase (GPAT) [E.C.→ Lysophosphatidic Acid2.3.1.15] esterifies 1st acyl-CoA to sn-1 position of(1-acyl-sn-glycerol 3-sn-glycerol 3-phosphatephosphate or “LPA”)LPA → Phosphatidic AcidLysophosphatidic acid acyltransferase (LPAAT) [E.C.(1,2-diacylglycerol2.3.1.51] esterifies 2nd acyl-CoA to sn-2 position ofphosphate or “PA”)LPAPA → 1,2-DiacylglycerolPhosphatidic acid phosphatase [E.C. 3.1.3.4](“DAG”)removes a phosphate from PA; DAG canOrsubsequently be converted to PC, PE orPA → Cytidine Diphosphatetriacylglycerols [“TAG”], wherein TAG synthesisDiacylglycerolrequires either a diacylglycerol acyltransferase(“CDP-DG”)(DGAT) [E.C. 2.3.1.20] or aphospholipid: diacylglycerol acyltransferase (PDAT)[E.C.2.3.1.158]CDP-diacylglycerol synthase [EC 2.7.7.41] causescondensation of PA and cytidine triphosphate, withelimination of pyrophosphate; CDP-DG can subsequentlybe converted to PI, PS, PG or CL
Following their de novo synthesis, glycerophospholipids can undergo rapid turnover of the fatty acyl composition at the sn-2 position. This “remodeling”, or “acyl editing”, is important for membrane structure and function, biological response to stress conditions, and manipulation of fatty acid composition and quantity in biotechnological applications. Specifically, the remodeling has been attributed to deacylation of the glycerophospholipid and subsequent reacylation of the resulting lysophospholipid.
In the Lands' cycle (Lands, W. E., J. Biol. Chem., 231:883-888 (1958)), remodeling occurs through the concerted action of a phospholipase, such as phospholipase A2, that releases fatty acids from the sn-2 position of phosphatidylcholine and acyl-CoA:lysophospholipid acyltransferases [“LPLATs”], such as lysophosphatidylcholine acyltransferase [“LPCAT”] that reacylates the Iysophosphatidylcholine [“LPC”] at the sn-2 position. Other glycerophospholipids can also be involved in the remodeling with their respective lysophospholipid acyltransferase activity, including LPLAT enzymes having lysophosphatidylethanolamine acyltransferase [“LPEAT”] activity, lysophosphatidylserine acyltransferase [“LPSAT”] activity, lysophosphatidylglycerol acyltransferase [“LPGAT”] activity and lysophosphatidylinositol acyltransferase [“LPIAT”] activity. In all cases, LPLATs are responsible for removing acyl-CoA fatty acids from the cellular acyl-CoA pool and acylating various lysophospholipid substrates at the sn-2 position in the phospholipid pool. Finally, LPLATs also include LPAAT enzymes that are involved in the de novo biosynthesis of PA from LPA.
Several recent reviews by Shindou et al. provide an overview of glycerophospholipid biosynthesis and the role of LPLATs (J. Biol. Chem., 284(1):1-5 (2009); J. Lipid Res., 50:S46-S51 (2009)). Numerous LPLATs have been reported in public and patent literature, based on a variety of conserved motifs.
The effect of LPLATs on polyunsaturated fatty acid [“PUFA”] production has also been contemplated, since fatty acid biosynthesis requires rapid exchange of acyl groups between the acyl-CoA pool and the phospholipid pool. Specifically, desaturations occur mainly at the sn-2 position of phospholipids, while elongation occurs in the acyl-CoA pool. For example, Example 16 of Intl. App. Pub. No. WO 2004/087902 (Renz et al.) describes the isolation of Mortierella alpina LPAAT-like proteins (encoded by the proteins of SEQ ID NO:31 and SEQ ID NO:33, having 417 amino acids in length or 389 amino acids in length, respectively) that are identical except for an N-terminal extension of 28 amino acid residues in SEQ ID NO:31. Intl. App. Pub. No. WO 2004/087902 also reports an increase in the efficiency of C18 to C20 elongation, an increase in Δ6 desaturation, and an increase in long-chain PUFA biosynthesis when one of these Mortierella alpina LPAAT-like proteins was expressed in an engineered strain of Saccharomyces cerevisiae that was fed exogenous 18:2 and α-linolenic [“ALA”; 18:3] fatty acids, that resulted in a large amount of the fatty acid substrates. Intl. App. Pub. No. WO 2004/087902 teaches that these improvements are due to reversible LPCAT activity in the LPAAT-like proteins and that not all LPAAT-like proteins have the LPCAT activity. Similar results were obtained upon expression of a LPCAT from Caenorhabditis elegans (clone T06E8.1) (Example 4 of Intl. App. Pub. No. WO 2004/087902; see also Intl. App. Pub. No. WO 2004/076617).
Numerous other references generally describe benefits of co-expressing LPLATs with PUFA biosynthetic genes, to increase the amount of a desired fatty acid in the oil of a transgenic organism, increase total oil content or selectively increase the content of desired fatty acids (e.g., Intl. App. Pub. Nos. WO 2004/076617, WO 2006/069936, WO 2006/052870, WO 2009/001315, WO 2009/014140).
Considerable efforts have focused on isolating LPLATs from the filamentous fungus, Mortierella alpina. In addition to the LPAAT proteins set forth as SEQ ID NO:31 and SEQ ID NO:33 (supra, isolated from Intl. App. Pub. No. WO 2004/087902), a variety of additional LPAAT homologs from Mortierella alpina have been described. For example, MaLPAAT3 (329 amino acids in length; SEQ ID NO:34 [SEQ ID NO:2 therein]) and MaLPAAT4 (313 amino acids in length; SEQ ID NO:35 [SEQ ID NO:4 therein]) are described in Intl. App. Pub. No. WO 2008/146745 (Suntory). U.S. Pat. No. 7,189,559 also describes a LPAAT homolog from Mortierella alpina of 308 amino acid residues (SEQ ID NO:37 [SEQ ID NO:2 therein]).
Despite the work described above, a novel LPAAT gene from the filamentous fungus Mortierella alpina is described herein. This gene is clearly differentiated from those M. alpina LPAAT-like sequences provided in the art and its expression has been demonstrated to improve the C18 to C20 elongation conversion efficiency, Δ4 desaturation conversion efficiency, and production of LC-PUFAs in oleaginous organisms expressing C18/20 elongase and Δ4 desaturase for synthesis of long-chain PUFAs.